As indicated in Scriver, C. R. et al "Metabolic Basis of Inherited Diseases" Vol. II (1995), Chap. 65, Inborn Errors in Bile Acid Biosynthesis and Storage of Sterols Other than Cholesterol, Bjorkhem, I. and Boberg, K. M., pp. 2073-2099, phytosterolemia (also referred to as sitosterolemia) is a rare inherited sterol storage disease involving increased intestinal absorption of phytosterol or shellfish sterols and decreased fecal secretion. It is characterized by "tendon and tuberous xanthomas and by a strong predisposition to premature coronary atherosclerosis . . . . Increased amounts of phytosterols (plant sterols), such as sitosterol and campesterol and their 5.alpha.-stanols, are found in blood, plasma, erythrocytes, and different tissues, especially in the xanthomas and arteries of affected subjects. Increased serum cholesterol and cholesterol have also been found in many patients." (p. 2073)
Patients afflicted with phytosterolemia have been found to have an increased incidence of coronary heart disease at an early age most likely due to early development of atherosclerosis at an early age. Bjorkhem et al, supra, indicate at page 2090 that "the mechanism behind the atherosclerosis is unexplained, but a high content of plant sterols in the circulating lipoproteins might promote their deposition in the arterial wall."
The microsomal triglyceride transfer protein (MTP) catalyzes the transport of triglyceride (TG), cholesteryl ester (CE), and phosphatidylcholine (PC) between small unilamellar vesicles (SUV). Wetterau & Zilversmit, Chem. Phys. Lipids 38, 205-22 (1985). When transfer rates are expressed as the percent of the donor lipid transferred per time, MTP expresses a distinct preference for neutral lipid transport (TG and CE), relative to phospholipid transport. The microsomal triglyceride transfer protein from bovine liver has been isolated and extensively characterized (1). This has led to the cloning of cDNA expressing the protein from several species, including humans (2). MTP is composed of two subunits. The small subunit is the previously characterized multifunctional protein, protein disulfide isomerase. This is supported by biochemical analysis of the protein (3) as well as co-expression studies performed in insect Sf9 cells using the baculovirus expression system. Expression of soluble active MTP requires the co-expression of PDI and the unique large subunit of MTP (4).
1: Wetterau, J. R. and Zilversmit, D. B. (1985) Chem. Phys. Lipids 38, 205-222. PA0 2. Sharp, D. et al, (1993) Nature 365, 65-69. PA0 3. Wetterau, J. R., et al, (1990) J. Biol. Chem. 265, 9800-9807. PA0 4. Ricci, B., et al, (1995) J. Biol. Chem. 270, 14281-14285. PA0 5. Wetterau, J. R., et al, (1992) Science 258, 999-1001.
Wetterau, J. R., et al, (1990) J. Biol. Chem. 265, 9800-9807.
Wetterau, J. R., et al, (1991) Biochemistry 30, 4406-4412.
Atzel, A., and Wetterau, J. R. (1993) Biochemistry 32, 10444-10450.
Atzel, A., and Wetterau, J. R. (1994) Biochemistry 33, 15382-15388.
Jamil, H., et al, (1995) J. Biol. Chem. 270, 6549-6554.
Lin, M. C. M., et al, J. Biol. Chem. 269, 29138-29145.
Nakamuta, M., et al, (1996) Genomics 33, 313-316.
Wetterau, J. R., et al, (1991) Biochemistry 30, 9728-9735.
In vitro, MTP catalyzes the transport of lipid molecules between phospholipid membranes. Presumably, it plays a similar role in vivo, and thus plays some role in lipid metabolism. The subcellular (lumen of the microsomal fraction) and tissue distribution (liver and intestine) of MTP have led to speculation that it plays a role in the assembly of plasma lipoproteins, as these are the sites of plasma lipoprotein assembly. Wetterau & Zilversmit, Biochem. Biophys. Acta 875, 610-7 (1986). The ability of MTP to catalyze the transport of TG between membranes is consistent with this hypothesis, and suggests that MTP may catalyze the transport of TG from its site of synthesis in the endoplasmic reticulum (ER) membrane to nascent lipoprotein particles within the lumen of the ER.
Abetalipoproteinemia is an autosomal recessive disease characterized by a virtual absence of plasma lipoproteins which contain apolipoprotein B (apoB). Kane & Havel in The Metabolic Basis of Inherited Disease, Sixth edition, 1139-64 (1989). Plasma TG levels may be as low as a few mg/dL, and they fail to rise after fat ingestion. Plasma cholesterol levels are often only 20-45 mg/dL. These abnormalities are the result of a genetic defect in the assembly and/or secretion of very low density lipoproteins (VLDL) in the liver and chylomicrons in the intestine. The molecular basis for this defect had not been previously determined. In subjects examined, triglyceride, phospholipid, and cholesterol synthesis appear normal. At autopsy, subjects are free of atherosclerosis. Schaefer et al., Clin. Chem. 34, B9-12 (1988). A link between the apoB gene and abetalipoproteinemia has been excluded in several families. Talmud et al., J. Clin. Invest. 82, 1803-6 (1988) and Huang et al., Am. J. Hum. Genet. 46, 1141-8 (1990).
Recent reports (5) demonstrate that the defect causing abetalipoproteinemia is in the MTP gene, and as a result, the MTP protein. When examined, individuals with abetalipoproteinemia have no MTP activity, as a result of mutations in the MTP gene, some of which have been characterized. These results indicate that MTP is required for the synthesis of apoB containing lipoproteins, such as VLDL, the precursor to LDL. It therefore follows that inhibitors of MTP would inhibit the synthesis of VLDL and LDL, thereby lowering VLDL levels, LDL levels, cholesterol levels, and triglyceride levels in animals and man.
Sharp, D., et al, (1993) Nature 365, 65-69.
Ricci, B., et al, (1995) J. Biol. Chem. 270, 14281-14285.
Shoulders, C. C., et al, (1993) Hum. Mol. Genetics 2, 2109-2116.
Narcisi, T. M. E., et al, (1995) Am. J. Hum. Genet. 57, 1298-1310.
Rehberg, E. F., et al, J. Biol. Chem (in press).
Canadian Patent Application No. 2,091,102 published Mar. 2, 1994 (corresponding to U.S. application Ser. No. 117,362, filed Sep. 3, 1993 (file DC21b)) which is incorporated herein by reference), reports MTP inhibitors which also block apoB containing lipoprotein secretion in a human hepatic cell line (HepG2 cells). This provides further support for the proposal that an MTP inhibitor would lower apoB containing lipoprotein and lipid levels in vivo. This Canadian patent application discloses a method for identifying the MTP inhibitors.
The use of microsomal triglyceride transfer protein (MTP) inhibitors for decreasing serum lipids including cholesterol and triglycerides and their use in treating atherosclerosis, obesity, hyperglycemia, and pancreatitis is disclosed in WO 96/26205, published Aug. 29, 1996, U.S. application Ser. No. 472,067, filed Jun. 6, 1995 (file DC21e), U.S. application Ser. No. 548,811, filed Jan. 11, 1996 (file DC21h), U.S. provisional application Ser. No. 60/017,224, filed May 9, 1996 (file HX79a*), U.S. provisional application Ser. No. 60/017,253, filed May 10, 1996 (file HX82*), U.S. provisional application Ser. No. 60/017,254, May 10, 1996 (file HX84*) and U.S. provisional application Ser. No. 60/028,216, filed Oct. 1, 1996 (file HX86*).
All of the above U.S. applications are incorporated herein by reference.